Ever since the discovery by Sir Alexander Fleming that the fungus Penicillium notatum produced a substance inhibitory to the growth of the human pathogen, Staphylococcus aureus, the reseach for antibiotics has consumed vast amounts of time and money.
Thousands of antibiotics produced by microorganisms have been identified. Most of them, unfortunately, are toxic not only to pathogens, but also to the human hosts that harbor the pathogens, and only a few dozen antibiotics have found their way into medical practice. The search continues for new antibiotics which will control particular pathogens, especially those which are resistant to known antibiotics. There is also a need for antibiotics which are effective against animal and even plant pathogens.
There are several classical methods for searching for and isolating microorganisms which produce antibiotics. In one method, soil is sprinkled onto the surface of a solid nutrient medium, such as gelled agar, in a petri dish and individual colonies of microorganisms are encouraged to grow. Circular areas clear of other growth around some of the colonies indicate that an antibiotic is inhibiting the growth of neighboring colonies. This is how antibiotic-producing colonies are identified. Each such colony must then be tested for its ability to inhibit the pathogen against which an antibiotic is sought. To test a colony, one may remove a plug of agar containing the active colony and place the plug on a second plate of nutrient medium which has been "seeded", or uniformly inoculated with the pathogen against which one seeks an antibiotic. A clear area surrounding the plug indicates that the antibiotic produced is effective against the pathogen.
This method has a shortcoming: For the antibiotic to be evident in the first place, it must be effective against neighboring bacteria cultured from, and present in, the original soil sample. Each antibiotic has its own particular spectrum of organisms against which it is effective. If an organism present in the soil sample produces an antibiotic effective against the pathogen, but not against neighboring soil-derived colonies, its effectiveness will be undetected. If an antibiotic producer is a slow grower, its neighbor may grow rapidly enough to overwhelm and mask the antibiotic effect. Most pathogens, being obligate parasites and many times fastidious, are often vulnerable to weak antibiotics, the very antibiotics least likely to be toxic to the pathogen host. Yet, it is the weak antibiotics which are most likely to be undetected by this method, for the reasons discussed.
A further shortcoming of this method is the time and space requirement for two-step culturing, first against neighboring colonies, and then against the pathogen. When thousands of tests are to be made these constraints are seriously limiting.
In another test method, an unknown sample of microorganisms, as for example from the soil, is grown directly upon a nutrient medium which has been preseeded with a pathogen, and clear areas are sought in the medium, indicating antibiotic action against the pathogen directly. In this case, it is required that the new antibiotic producer thrive in the same environment as the pathogen - at the same temperature, pH, aerobicity, and so forth. Pathogens, however, frequently require exotic media and often require a special pH or redox potential which may not be acceptable to the culture of the very microorganisms which produce the sought-for antibiotic. There is, therefore, an even larger risk of passing up the most promising new candidate microorganisms. In this method, temperature is a major problem since most human pathogens require a temperature of 37.degree. C. and most soil microorganisms grow best at about 25.degree. C. Additionally, if the pathogen is a strict anaerobe (cannot grow in the presence of oxygen), aerobic antibiotic-producing organisms effective against the pathogen will not be found.
As indicated previously, soil is the source material for almost all antibiotic-producing organisms. The only practical way to find new antibiotics is to conduct massive soil screening programs. The methods described above, and with minor permutations thereof, have been employed since the search for antibiotic-producing microorganisms began. Many potentially valuable antibiotics have surely been missed in these screening procedures, since the procedures rely on the ability of the antibiotic-producing microorganism either to inhibit its neighbors, or to produce an antibiotic when cultured under conditions optimal for the growth of the pathogen, rather than optimal for the antibiotic-producing microorganism. Under these constraints, the cost of finding a new antibiotic-producing organism of potential value is becoming economically impractical. There is, then, an urgent need for an effective and efficient method for screening source materials (e.g. soils) for microorganisms capable of inhibiting specific pathogens.
This invention is a unique device and method for culturing microorganisms which fills this need. Two cultures, e.g. a pathogen and mixed soil microorganisms, are grown sequentially in the same vessel, each on its preferred medium. The two media in the vessel are disposed, sandwich-like, back-to-back. Antibiotics produced on one side of the media sandwich diffuse through it and inhibit growth (cause a clear area) on the pathogen side of the sandwich. Even though an antibiotic is too weak to inhibit neighbor colonies, it will inhibit the pathogen growing on the other side of the media sandwich. Even though an antibiotic producer grows more slowly than its neighbors, the antibiotic action on the pathogen is evident.